African Green Monkey kidney epithelial cells (Vero) were maintained in complete Dulbecco’s modified Eagle’s medium (cDMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine and 4% antibiotic-antimycotic mixture. Primary human brain microvascular endothelial cells (HBMECs) were used at passage 14 and were grown in complete RPMI-1640 Medium (cRPMI) supplemented with 20% heat inactivated FBS, 2mM L-glutamine, 1mM Sodium Pyruvate, 1% MEM non-essential amino acids, 1% MEM vitamins and 5ml penicillin/streptomycin. When cells were confluent they were harvested with trypsin-EDTA and passaged at a sub-cultivation ratio of 1:3 into new culture flasks with fresh corresponding medium (cDMEM for Vero cells and cRPMI for HBMECs). Cells were considered confluent when their expansion had reached a point where cells touched each other on all sides and no intercellular gaps could be observed. K562 cells, a human erythroleukemia cell were cultured in cRPMI medium supplemented with L-glutamine, 10% FBS and antibiotic–antimycotic mixture, and sub-cultured twice a week at initial concentration of 105 cells ml-1. All cells lines were grown at 37°C under humidified 5% CO2 conditions. To exclude if cell viability could be regarded as a factor affecting parasite interaction with host cells and therefore any subsequent measurements, viability of cells was assessed on a minimum of 200 cells using the trypan blue exclusion assay prior to inoculation onto cultured flasks. Cell viability was greater than 95% at all times. Parasite viability was also checked using the Alamar Blue assay and only those greater than 95% were used. Vero cells and K562 cells were obtained from the European Collection of Animal Cell Cultures (Porton Down, Salisbury, Wiltshire, UK). All media, FBS, tissue culture supplements and antibiotics were from GIBCO. HBMECs were originally obtained from ScienCell Research Laboratories.
Neospora caninum (Nc-Liverpool) strain was obtained from Professor S. Trees (University of Liverpool) and was propagated in Vero cells as described . Parasites were harvested from their feeder cell culture and purified as described previously . The number of tachyzoites was estimated using a haemocytometer. The final volume of suspension was adjusted with culture medium to achieve a ratio of 1:1 parasite/host cell for subsequent infection experiments. Parasite viability was also checked using Alamar Blue assay and only those greater than 95% were used. All experiments were conducted at least in triplicate.
N. caninum infection of HBMECs
HBMECs were grown on poly-L-lysine coated coverslips in 6-well cell culture plates and infected with N. caninum tachyzoites at a host-parasite ratio of 1:1. For controls, non-infected cells were sham inoculated with an equivalent volume of media without tachyzoites. After 1 hr of initial incubation, the media from the wells was removed and fresh cRPMI added. K562 cells were infected as described previously .
Time-course of infection by N. caninum
To monitor the proliferation of tachyzoites within the host cell, cells were fixed in cold acetone:methanol (1:1) for 10 min at 1, 3, 6, 12, 24 and 48 hr post infection (pi). Infected cells and controls were processed for immunofluorescent staining as described . Briefly, fixed cells were blocked in 10% FBS for 1 hr at ambient temperature and incubated with primary monoclonal mouse antibodies against anti-NcSAG1 (a kind gift from Professor Andrew Hemphill, University of Bern, Switzerland) in 1:400 dilution for 2 hr at ambient temperature. Alexa Fluor 488 (FITC filters) conjugated goat anti-mouse secondary antibodies (Invitrogen Ltd, Paisley, UK) at 1:500 dilution in TRIS buffered saline (TBS), were used to detect bound primary antibody by immunofluorescence after 2 hr incubation in the dark at ambient temperature. The coverslips were carefully removed from the cell culture plates and were mounted with ProLong Gold antifade reagent with 4′,6-diamino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes, Inc., Eugene, OR) on microscopic slides. Images were captured on a Leica microscope (Leica Microsystems Imaging Solutions Ltd, Cambridge, UK). Because of the inherent subjectivity of the qualitative analysis, quantitative measurements of the surface area of the parasite were made by counting pixels or groups of pixels that were contributed by parasites. A region of interest from a large fluorescent image was selected and a threshold value for detection visually obtained from the green colour intensity of the parasites. The total number of pixels was counted automatically based on the green colour intensity above the threshold value. Using the scale bar on the image, the corresponding size and area of each pixel, and therefore, the surface area of parasites were calculated. The mean surface area ± standard deviation of the parasitic structure was measured per time points. Measurement of the parasite surface area was performed three times and are quoted in arbitrary units.
To assess the gross kinetics of N. caninum in HBMECs cells were cultured in 75cm2 culture flasks and grown in cRPMI. Once a confluent monolayer of cell was formed, parasites were added at a host-parasite ratio of 1:1. Flasks were viewed under the microscope every 24 hr and the number of parasitic lysis areas in 10 microscopic fields at x 10 magnification was recorded and the average for the flask was calculated. This experiment was repeated three times. Data is shown as average ± standard deviation.
Detection of mitochondrial integrity and membrane potential (ΔΨm)
Mitochondrial integrity and membrane potential (ΔΨm) in normal and infected HBMECs was determined using the DePsipher kit (Assay Designs, USA) following the manufacturer’s instructions. This kit uses a unique cationic dye to indicate the loss of the mitochondrial potential. In cells with disrupted mitochondrial potential, the dye remains in the cytoplasm as a green fluorescent monomeric form, whereas in bioenergetic healthy cells the dye appears red following aggregation of the DePsipher dye within the mitochondria.
HBMECs were grown on coverslips as described above. Cells were infected with N. caninum tachyzoites at a host-parasite ratio of 1:1. Cells were stained at 1, 3, 6, 12, 24 and 48 hr pi. At each time point, the medium was discarded and cells were washed with 1ml pre-warmed (37°C) 1X diluted reaction buffer with stabilizer solution. Supernatant from diluted DePsipher solution centrifuged at 13,000 x g for 1 min at ambient temperature was added to the cells and they were then incubated for 30 min. Cells were washed again with 1ml pre-warmed 1X reaction buffer with stabilizer. The coverslip was then mounted onto a microscopic slide and viewed under the microscope immediately. The green monomers (Ex485nm/Em/535nm) and red aggregates (Ex560nm/Em595nm, a sensitive marker of ΔΨm) were monitored by fluorescence microscopy. Mitochondrial staining was repeated three independent times.
Cell metabolism assays
To assess changes in host cell viability and bioenergetics following N. caninum infection, two different methods were employed. First, the colorimetric MTT assay [CellTiter 96 Non-Radioactive Cell Proliferation Assay; Promega, UK], which is based upon formazan production due to reduction by cellular reducing equivalents, was used. Although the MTT assay is usually utilized to monitor cell-proliferation, the fact that it depends on the cellular redox state means that it is essentially a measurement of total biological reduction potential within the assay volume . The MTT assay was performed following the manufacturer’s instructions. Briefly, HBMECs were seeded in 96-well culture plates at 104 cells per well and grown in cRPMI at 37°C in a humidified atmosphere containing 5% CO2. When the cells were confluent, tachyzoites of N. caninum were added at a host-parasite ratio of 1:1. Non-infected controls were sham inoculated with an equal volume of medium without tachyzoites. After 1 hr of initial incubation, medium from the wells was removed and fresh cRPMI was added. At 1, 2, 3, 6, 12, and 24 hr pi, 15μl of MTT dye solution was added to each well. Following incubation for 4 hr, 100μl of solubilisation solution/stop mix was added and incubated for a further 1 hr. Absorbance at 590 nm was recorded using a Multiskan Ascent plate reader [Labsystems]. This experiment was repeated at least 3 times.
The second method to assay changes in cellular bioenergetics was by measurement of the oxygen consumption rate [OCR] which results directly from mitochondrial oxidative respiration as previously described . Briefly, the OCR of suspensions of free parasites, suspensions of infected or non-infected cell monolayers was measured polarographically using Clark Oxygen Electrodes [Rank Brothers, Bottisham, UK]. Suspensions of cells, volume 1 ml, were continuously stirred in sealed electrode chambers at 37°C. Since this is a closed system, the O2 consumed during oxidative respiration cannot be replaced from the air so the concentration of dissolved O2, [O2aq falls. [O2aq was measured at a polarographic voltage of −0.6 V. Electrodes were calibrated with a two point method in accord with the manufacturer’s instructions: 100% saturation with air [[O2]aq ≈ 0.25 mM at 37°C] and 0% O2 by the addition of a stoichiometric excess of Na2S2O4, a strong reducing agent. These experiments were performed in a HEPES-buffered Hanks solution which contained (in mM): 138 NaCl, 4.2 NaHCO3, 1.2 NaH2PO4, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 10 HEPES (pH 7.4 with NaOH) and 0.1% fatty acid free BSA. Since the steady-state rates of oxygen consumption [OCR] were linear, OCR was taken as the slope in [O2aq over 3–5 min epoch. After a 10 min equilibration period, the OCR was measured first in the absence of exogenous substrate (0 glucose), then in the presence of 10mM glucose (by addition of 10μl of a 1M glucose stock solution in H2O to the 1ml incubation volume), then in the continued presence of 10mM glucose but with the addition of the electron donor shuttle pair: 0.2 mM tetramethyl-p-phenylenediamine with 1 mM ascorbate TMPD; . To determine the OCR that arose solely through oxidative respiration, the mitochondrial reduction of O2 was then blocked by the addition of 6 mM NaN3 to abolish cytochrome c oxidase activity. To determine whether the cells, naïve or infected, or the free parasites themselves, preferentially metabolised monocarboxylic acids compared to glucose, their ability to respire L-lactate and methyl-pyruvate, a membrane permeable analogue of pyruvate, was tested. OCR was measured as described earlier, but with glucose substituted for by an equimolar amount of either L-lactate (by addition of 10μl from a 1M L-NaLactate stock solution, made in H2O, to the 1ml incubation volume) or methyl-pyruvate, a membrane permeable analogue of pyruvate (again by addition of 10μl from a freshly made 1M stock solution, made with H2O, to the incubation volume). Since lactate was without effect, no control was deemed necessary for the associated change in Na+ concentration: 138 to 148mM that occurred on addition of the lactate salt.
To control for O2 consumption that was either due to the background consumption by the electrode, which was non-mitochondrial in origin, or which arose due to autoxidation of ascorbate by atmospheric oxygen, all OCRs were corrected by subtraction of the OCR measured under identical experimental conditions in the absence of biological tissue or/and after the addition of NaN3. Each separate experiment constituted a biological replicate, and contributes to the experimental number: n.
Given the difference in cellular scale between tachyzoites and HBMECs, in order to compare OCR between the two different host cell types and free parasites we attempted to scale them by the ratio of their DNA content. It is well documented that the DNA content of cells at least in vertebrates is highly correlated and proportional to cell size . It was assumed that this assumption also held true for the apicomplexan protozoa even though they have extra-nuclear DNA that is non-mitochondrial in origin. Normalization of OCR with DNA content does, however, allow for subsequent comparison to further studies and the literature. Total genomic DNA was extracted from 1 × 106 of each of N. caninum tachyzoites, HBMECs or K562 cells, of similar density, using DNeasy Tissue kit (Qiagen, Valencia, California) following the manufacturer’s instructions. The extracted DNA was quantified by using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE). The ratio of DNA content relative to that in free tachyzoites was 6.44 for HBMECs and 8.6 for K562 cells; the DNA content being 10.3 pg μl-1 for HBMECs, 13.8 pg μl-1 for K562 cells, and 1.6 pg μl-1 for the tachyzoites.
All data was checked for normality using the D’Agostino & Pearson omnibus normality test. Data is presented and analysed in a form appropriate to its distribution, with the test used stated in brackets. Parametric statistical analysis using PRISM (5.03, GraphPad Software, San Diego California, USA) were performed if data was deemed to be normally distributed, otherwise non-parametric analyses were performed with StatsDirect (StatsDirect Ltd, Altrincham, Cheshire, UK). Tukey’s Multiple Comparison Test was used for multiple comparisons across groups. When non-parametric data was compared with Kruskal-Wallis Conver-Inman , all groups were compared to each other, with only the specific comparisons quoted. Since the effects of glucose and TMPD/ascorbate within a given data group, as identified by host cell-type and state of infection (non-infected, 1 hr or 24 hr p.i.), were repeated measurements, the Friedman test was performed with all pairwise comparisons according to Conover . All P values were two-tailed and adjusted for multiple comparisons when necessary using Bonferonni correction. P < 0.05 was considered statistically significant. The statistical test used, the P obtained when significant and n, the number of data determinations are given in parentheses.